Pan-cancer and multi-omics analyses revealed the diagnostic and prognostic value of BAZ2A in liver cancer

BAZ2A, an epigenetic regulatory factor that affects ribosomal RNA transcription, has been shown to be highly expressed in several cancers and promotes tumor cell migration. This study explored the expression and mechanism of BAZ2A in tumorigenesis at the pan-cancer level. The Cancer Genome Atlas, Gene Expression Omnibus databases and TIMER2.0, cBioPortal and other tools were used to analyze the level of expression of BAZ2A in various tumor tissues and to examine the relationship between BAZ2A and survival, prognosis, mutation and immune invasion. In vitro experiments were performed to assess the function of BAZ2A in cancer cells. Using combined transcriptome and proteome analysis, we examined the possible mechanism of BAZ2A in tumors. BAZ2A exhibited high expression levels in multiple tumor tissues and displayed a significant association with cancer patient prognosis. The main type of BAZ2A genetic variation in cancer is gene mutation. Downregulation of BAZ2A inhibited proliferation, migration, and invasion and promoted apoptosis in LM6 liver cancer cell. The mechanism of BAZ2A in cancer development may involve lipid metabolism. These results help expand our understanding of BAZ2A in tumorigenesis and development and suggest BAZ2A may serve as a prognostic and diagnostic factor in several cancers.


BAZ2A phosphorylation in pan-cancer
Using the CPTAC dataset, we analyzed the levels of BAZ2A phosphorylation in four cancer types (LIHC, KIRC, OV, and HNSC).The S1395 site of BAZ2A showed higher phosphorylation levels in LIHC and KIRC tumor tissues in comparison to normal tissues, but lower phosphorylation levels in OV tumor tissues (Supplementary Fig. 1B).S1768 phosphorylation levels were also higher in LIHC, OV, HNSC tumor tissues compared with normal tissues.Our results also revealed several sites with increased phosphorylation in HNSC tumor tissues, including S115, S494, S1768, Y1775, and S1776.These findings imply that alterations in the post-translational modifications of BAZ2A might contribute to the process of tumorigenesis.

The association between BAZ2A expression and tumor diagnosis and prognosis
Subsequently, we used the TCGA dataset to examine the association between BAZ2A expression and patient outcomes in 21 different cancers.Figure 2A,B showed statistically significant cancer types.The survival analysis indicated that increased expression of BAZ2A was linked to a poor prognosis in patients with LIHC, KIRP, pheochromocytoma and paraganglioma (PCPG), and endometrial cancer (UCEC), while reduced expression of BAZ2A was linked to an adverse prognosis in KIRC (Fig. 2A,B).
ROC curves were employed to assess the diagnostic utility of BAZ2A across multiple cancer types.BAZ2A showed some diagnostic accuracy for LIHC (0.846), KIRC (0.642), PCPG (0.741), and UCEC (0.61) (Fig. 2C).These results showed that BAZ2A expression influenced prognosis in several cancers and that BAZ2A gene expression may serve as a diagnostic marker in some cancers.

Genetic alterations are correlated with DNA methylation of the BAZ2A promoter
We used two probes (cg12199011, cg20829193) to analyze TCGA RNAseq and Methylation450 data to detect DNA methylation levels of the BAZ2A gene promoter.The findings demonstrated that BAZ2A expression was inversely linked to promoter methylation in LIHC, KIRC, KIRP, PCPG, and UCEC (Fig. 3A).
Analysis of the gene alteration frequency revealed a variety of genetic variations in the BAZ2A gene in cancer, such as mutations, structural variations, amplification, and deletions, among which mutation was the most frequently detected variation, including missense mutation, truncating mutation, inframe mutation, splice mutation, fusion mutation (Fig. 3B,C).Cutaneous melanoma (SKCM) exhibited the highest frequency of BAZ2A gene alteration (> 10%) (Fig. 3B).The sites and types of BAZ2A gene alterations are shown in Fig. 3C.Missense mutation were the major categories of genetic alterations; the R553C mutation was detected in three UCEC samples and one SKCM sample (Fig. 3C,D).
Survival analysis indicated that patients with BAZ2A gene mutations had a poorer prognosis in terms of OS, PFS, DFS, DSS, and DSS in LIHC and KIRC than patients without BAZ2A gene mutation (Fig. 3E,F).In    www.nature.com/scientificreports/UCEC, the OS, PFS, DSS of patients with BAZ2A gene mutations were better than those without BAZ2A gene mutation (Fig. 3G).

Association of BAZ2A gene expression and immune infiltration
Next, we used a timer platform to assess the relationship between BAZ2A gene expression and the degree of immune cell infiltration in 31 cancers.In most cancers, including LIHC, KIRC, KIRP, PCPG, and HNSC (including HNSC+HPV−, HNSC+HPV+), we observed a positive correlation between BAZ2A mRNA expression and the levels of Tregs and macrophage cell infiltration (Fig. 4A).The scatterplots generated by the TIMER also demonstrated that BAZ2A expression exhibited a association with the level of invasion of immune cells in different tumors (Fig. 4B).Taken together, BAZ2A gene expression is positively correlated with immune cell infiltration in multiple cancer types.

Enrichment analysis of proteins and genes associated with BAZ2A
Next, we conducted a screening for BAZ2A interacting proteins and genes, followed by enrichment analyses.Through the utilization of the STRING database, The amount is 31 BAZ2A-interacting proteins were identified, and the network of interactions among these proteins is visualized in Fig. 5A.BAZ2A-related genes with the highest enrichment in biological process (BP), cellular component (CC), and molecular function (MF) are shown in Fig. 5B.The KEGG analysis revealed a markedly enrichment of BAZ2A-related genes in the lysine degradation pathway, the TGF-β signaling pathway, and viral life cycle (Fig. 5B,C).Analysis of RNAseq data from TCGA showed that BAZ2A and its related genes, KMT2D, CELF1, CCNT1, CREBBP, SMG1, and SRCAP genes, were co-expressed in LIHC, KIRC, KIRP, PCPG, and UCEC (Fig. 5D).

BAZ2A is highly expressed in LIHC and promotes the malignant behavior of LIHC cells
Through the analysis of various cancers, we found that the high expression of BAZ2A has significant effects on the survival, prognosis, immune invasion and other aspects of LIHC, while it only has significant effects on some aspects of other cancers.Therefore, we chose LIHC to further explore the role of BAZ2A in cancer.We selected LIHC to further explore the function of BAZ2A in cancer.We examined several hepatoma cell lines and found that that BAZ2A transcription levels were highest in LM6 cells (Fig. 6A).Thus, we selected this cell line for subsequent experiments.We designed siRNAs against BAZ2A and confirmed their efficacy (Fig. 6B).CCK8 and clonal formation experiments showed that BAZ2A downregulation inhibited cell proliferation and clonal formation of LM6 cells (Fig. 6C,D).Transwell experiments showed that BAZ2A silencing reduced cancer cell migration and invasion (Fig. 6E,F).Flow cytometry showed that downregulation of BAZ2A led to increased cell apoptosis, which was supported by western blot of Bax and other apoptosis-related proteins (Fig. 6G,H).The function of BAZ2A in facilitating cancer cell migration was further validated by the detection of epithelial-mesenchymal transition (EMT)-related molecules (Fig. 6I).
We collected tumor tissue samples from 80 patients with liver cancer and examined BAZ2A expression by immunohistochemistry. BAZ1A was highly expressed in liver cancer tissues relative to the adjacent cancer tissues in 30 samples (Fig. 6J,K).

Multiomics analysis of BAZ2A
To further validate the relationship between BAZ2A and cancer, we performed transcriptome and proteome sequencing in cells silenced for BAZ2A or transfected with NC siRNA as a negative control.The correlation analysis of transcriptomics and proteomics results indicated that upon the downregulation of BAZ2A expression, the numbers of proteins and genes with significant differences were associated, and the correlation between differential proteins and differential genes was good (Fig. 7A,B).
The differentially expressed proteins identified by proteome sequencing were subjected to analysis using GO and KEGG.GO bubble plots indicated that most of the differential proteins enriched in BP were related to lipid metabolism processes, such as cellular lipid catabolic process and triglyceride catabolic process (Fig. 7C).The differential proteins enriched in MF were related to lipoprotein receptor activity and lipoenzyme activity, such as triglyceride lipase activity and lipoprotein particle receptor activity (Fig. 7D).The differential proteins enriched in CC were associated with lipid components, including the membrane part and lipoprotein particle (Fig. 7E).These results are illustrated by directed acyclic plots in Supplementary Fig. 2A-C.
Similar results were obtained from the combined proteome and transcriptome analysis.In GO analyses with significant enrichment in both omics, BP differential genes and differential proteins were also enriched in lipid metabolism processes, with 34 proteins and 328 genes enriched in the lipid catabolic process.CC-enriched differential proteins and differential genes were associated with lipid components, with 70 proteins and 1196 genes enriched in the intrinsic component of membrane (Fig. 7F).The findings obtained were supported by transcriptome KEGG enrichment, in which the highest degree of enrichment was in the metabolic pathway (Fig. 7G).These findings support a role for BAZ2A in lipid metabolism processes.
Figure 7G shows the PPI network of the 73 differential proteins; the core protein is SOX2 (Fig. 7H).Abnormal expression of SOX2 was shown to be intricately linked to the occurrence, differentiation, metastasis, and poor prognosis of malignant tumors 19,20 .We performed KEGG enrichment analysis on the 73 differential proteins and observed that these DEPs were enriched in multiple signaling pathways of cancer, such as the RAS, MAPK signaling pathway, PI3K-AKT signaling pathway, and Hippo signaling pathway (Fig. 7I).This suggests that BAZ2A may be involved in the cancer-related processes by regulating key proteins and molecules involved in these processes.

Discussion
Pan-cancer analysis involves the utilization of diverse databases to analyze information such as gene expression, prognosis, and mutation in different cancers; these findings are critical to provide new insights into tumor prevention and personalized treatment options 21 .Using multiple databases and tissue chip analysis, we explored the pan-cancer expression profile of BAZ2A and its potential association with prognosis and immune invasion in cancer patients.Our study suggests that BAZ2A functions as a promoter of cancer that may participate in cancer www.nature.com/scientificreports/progression and promote tumor malignant behavior by regulating lipid metabolism.The results of our functional experiments in hepatoma cells further substantiate the function of BAZ2A in facilitating cancer cell proliferation.Apart from alterations in BAZ2A expression observed in cancer, we also found that the BAZ2A gene is mutated in variety of tumor types and BAZ2A protein also exhibited changes in phosphorylation levels.Gene mutations 22,23 and phosphorylation 24 are common mechanisms to affect protein activity.Therefore, we speculate that the role of BAZ2A on tumorigenesis and development is not only regulated by changes in its protein levels but it may also be regulated by mutations and phosphorylation in cancer cells.
The survival analysis demonstrated that high expression of BAZ2A was associated with adverse prognosis in patients with tumors such as LIHC, but low expression of BAZ2A was associated with adverse prognosis in KIRC.This suggests that the function of BAZ2A in cancer may be tumor-specific, and the mechanism by which it functions in different tumors may be complex, involving different signaling pathways and related molecules.To explore its mechanism of action, we examined the transcriptome and proteome associated with BAZ2A in hepatoma cells.We also analyzed the transcriptome and metabolome of BAZ2A in other tumors, such as cervical cancer.In subsequent studies, we will further analyze the unique and common mechanisms of BAZ2A in different tumors.www.nature.com/scientificreports/DNA methylation is a common mechanism of regulating gene expression [25][26][27] .We found that in a variety of cancers, the increased expression of BAZ2A was found to be correlated with DNA methylation occurring in its promoter region.
Considering the crucial role of the tumor microenvironment on tumorigenesis and development [28][29][30][31][32][33][34] , the effect of BAZ2A on immune cell infiltration was analyzed.Our findings demonstrated that BAZ2A expression may be related to Treg and macrophage cell infiltration in a variety of cancers and suggested that BAZ2A may participate in tumor development by interacting with a variety of immune cells.
Our analysis revealed that BAZ2A exhibited elevated expression levels in tumor tissue from liver cancer patients in only 30 of 80 cases; this finding may be the small number of samples.Further investigations with a larger sample size are necessary to more comprehensively assess the expression of BAZ2A in cancer tissues and its potential correlation with various indicators.
Our results also indicated that BAZ2A may participate in the lipid metabolism process of cells.Thus, we speculate that BAZ2A may affect cell proliferation and migration through metabolic reprogramming, which may influence the occurrence and development of tumors.

Conclusion
Our study shows that BAZ2A expression correlates with prognosis and may be a potential diagnostic marker in several cancers.BAZ2A has been implicated in facilitating tumor cell proliferation, migration, and epithelialmesenchymal transition (EMT), while exerting inhibitory effects on apoptosis in tumor cells.Taken together, these observations suggest that BAZ2A may present as a promising therapeutic target for multiple cancer types.

Analysis of BAZ2A expression in tumor
BAZ2A gene expression was examined in data from The Cancer Genome Atlas (TCGA) using TIMER2.0 35(http:// timer.cistr ome.org/).R (version 3.6.3)and the "ggplot2" package in R were utilized for the analysis and visualization of RNAseq data.The Clinical Proteomic Tumor Analysis Consortium (CPTAC) dataset of UALCAN was employed to explore the correlation between BAZ2A expression and the pathological stage of various cancers.To investigate the variations in BAZ2A phosphorylation levels between cancerous and non-cancerous tissues, an analysis of data sourced from the UALCAN (http:// ualcan.path.uab.edu/ analy sis-prot.html) was conducted utilizing CPTAC methods 36 .

Survival analysis and ROC curve analysis
Kaplan-Meier 37 was performed to evaluate the influence of BAZ2A expression on overall survival (OS) and disease-specific survival (DSS) (https:// kmplot.com/ analy sis/).ROC curve analysis of RNAseq data from TCGA and GTEx was utilized to assess the significance of BAZ2A in cancer diagnosis using the R package "pROC" package and "ggplot2" to calculate the area under the curve (AUC).

DNA methylation and mutation analysis
R language and the "ggplot2" package were used for analysis and visualization of data.Two probes of the BAZ2A promoter (cg12199011, cg20829193) were selected to detect its DNA methylation level.
The "Quick Search" module of the cBioPortal database was employed to select the analysis function of TCGA pan-cancer atlas, obtain the Mutation information of BAZ2A gene, and "Mutations" are used to obtain the specific mutation site information on the BAZ2A functional domain map, Click "The view of 3 D Structure" can get a 3 D model diagram (https:// www.cbiop ortal.org/) 38 .We used the "Comparison" module of cBioPortal to analyze the clinical outcomes of BAZ2A gene mutation in LIHC, KIRC and UCEC, including OS, DSS, progression-free survival (PFS), and disease-free survival (DFS).

Immune infiltration analysis
The connection between BAZ2A expression and cancer-associated fibroblasts in different tumor types in TCGA database was investigated through the immune association (gene) module in the TIMER 2.0 database (http:// timer.cistr ome.org/).

Enrichment analysis
The protein-protein interaction network of BAZ2A was obtained using the STRING database 39 (http:// string.embl.de/).Parameter settings were as follows: max number of interactors to show: (custom value, max interactors: 30); minimum required interaction score: [Low confidence (0.150)]; meaning of network edges: (evidence); the active interaction sources: (textmining, experiments, co-expression, neighborhood, co-occurrence).The "Similar Gene Detection" module of GEPIA 2.0 was employed for the purpose of obtaining the top 100 BAZ2A-related genes by comparing the differential genes between tumor and normal tissues contained in all TCGA databases.Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) 40 data were analyzed by R packages "ggplot2" and "clusterProfiler." Co-expression analysis of BAZ2A, KMT2D, CELF1, CCNT1, CREBBP, SMG1, and SRCAP was performed using https:// portal.gdc.cancer.gov/.

Figure 2 .
Figure 2. Cancer prognostic and diagnostic analyses based on BAZ2A expression.(A) The relationship between BAZ2A mRNA expression and overall survival in TCGA and GEO datasets.P < 0.05 is considered as a statistical difference.(B) The relationship between BAZ2A mRNA expression and disease-free survival was evaluated using the TCGA dataset.P < 0.05 is considered as a statistical difference.(C) ROC analysis of cancer diagnosis based on BAZ2A mRNA expression levels.

Figure 3 .
Figure 3.DNA methylation analysis and characterization of BAZ2A genetic variations in cancer.(A) BAZ2A promoter methylation levels correlated with BAZ2A mRNA levels in LIHC, KIRC, KIRP, PCPG, and UCEC.P < 0.05 is considered as a statistical difference.(B) The cBioPortal database was used to examine BAZ2A mutations in pan-cancer, and the frequency of different types of BAZ2A mutations in each cancer is shown.(C) The mutation sites and mutation types of BAZ2A gene variations in cancer and the corresponding number of cases in TCGA.(D) The three-dimensional structure of BAZ2A and the most frequently mutated site (R553C) in cancer.(E-G) We employed the cBioPortal database to study the effects of BAZ2A mutation status on various survival outcomes, including overall survival, disease-free survival, progression-free survival, and diseasespecific survival in LIHC (E), KIRC (F), and UCEC (G).P < 0.05 is considered as a statistical difference.

Figure 4 .
Figure 4. Correlation analysis of BAZ2A expression and immune cell infiltration in cancer.(A) Correlation between BAZ2A expression and infiltration of T regulatory cells and macrophages using the TIMER2.0database.P < 0.05 is considered as a statistical difference.(B) Correlation of BAZ2A expression with levels of the infiltration of immune cells in LIHC, KIRC, KIRP, PCPG, and UCEC.P < 0.05 is considered as a statistical difference.

Figure 5 .Figure 7 .
Figure 5. Enrichment analysis of BAZ2A-associated proteins and genes.(A) BAZ2A-interacting proteins were obtained using the STRING database and shown by the STRING protein network diagram.(B) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of BAZ2A and its related genes.P < 0.05 is considered as a statistical difference.(C) Analysis of the enrichment pathways of BAZ2A-related genes.(D) Co-expression analysis of BAZ2A and associated genes in LIHC, KIRC, KIRP, PCPG and UCEC (https:// www.xiant ao.love/).*P < 0.05, **P < 0.005, ***P < 0.001.P < 0.05 is considered as a statistical difference.

Table 1 .
Screening process of cancer species.⭐ ⭐⭐⭐ P < 0.001.P < 0.05 is considered as a statistical difference; The abbreviation "ns" indicates non-significant results.//: There is no sample data for this cancer in the corresponding database/this cancer type is not used.